ssDNA phage FLiP resides in dsDNA form in resistant Flavobacterium host

Bacteria in the genus Flavobacterium play a key role in organic matter decomposition in the aquatic environment. Phages infecting these bacteria regulate the host populations and thereby the ecosystem functions. However, bacterial resistance against phage may cause changes in bacterial phenotypic characteristics. The single stranded DNA phage Finnlakevirus FLiP infects three known environmental Flavobacterium sp. isolates: B330, B167, and B114. Building on our previous FLiP-host interaction studies, we investigated resistance mechanisms from the host perspective, aiming to understand how Flavobacterium strains resist FLiP and related phages. We assessed the fitness effects of phage resistance by comparing growth dynamics between ancestral and resistant variants. In the absence of phage, no significant differences in growth was observed, indicating that resistance to single stranded DNA phage FLiP does not impose a detectable fitness cost under laboratory conditions. Next, we screened host genomes for anti-phage systems and compared host genomes across strains and between ancestral and resistant variants. Genomic comparisons revealed resistance-related mutations, although no single mutation or anti-phage system consistently explained FLiP resistance across strains. Furthermore, we evaluated the possibility of lysogeny and superinfection immunity using sequence analysis, PCR, and nuclease treatments. Notably, resistant B114 host harbored the FLiP genome as a circular extrachromosomal double stranded DNA element, suggesting potential for lysogeny. Surprisingly, low levels of FLiP sequences were detected in bacterial populations not exposed to FLiP in the laboratory. Our findings suggest that FLiP-type phages may persist in host populations as extrachromosomal double stranded DNA elements in a subset of cells. This strategy could allow phages to endure unfavorable conditions and regulate infection timing. As detected in previous experiments, rather than requiring optimal conditions, FLiP is capable of productive infection even under stress, with infection stalling only when host growth is severely limited. The constant persistence within the host population, and capability to start particle production as soon as conditions improve, may represent an evolutionary adaptation for survival and transmission in fluctuating environments.